S. pneumoniae (Sp) colonizes the nasopharynx of up to 90% of children leading to 1M deaths per year from pneumococcal diseases (PD). Near half colonized children will carry at least two Sp strains in the nasopharynx. Whereas the recent worldwide rollout of pneumococcal conjugate vaccines has reduced the global burden of PD, its impact on nasopharyngeal (NP) carriage is hampered by a lack of knowledge about population dynamics of Sp strains in the human nasopharynx. Sp biofilms (SPB) are implicated in colonization of, and persistence in, the nasopharynx; however, we know virtually nothing about the behavior of Sp strains in their natural niche. Furthermore, since humans are the sole host for Sp, recombination of Sp strains in the nasopharynx should be the way multi-drug resistant bacteria and perhaps new serotypes arise. Therefore, this proposal will investigate colonization and persistence (population dynamics) of pairwise of clinically-important vaccine type strains when SPB forms on human pharyngeal cells; the structure of these biofilm consortiums will be studied by microscopy. We will also begin investigating the mechanism of recombination within Sp biofilm consortiums. In specific Aim#1 all possible pairwise of three clinically-important vaccine type strains, serotype 4, 6B, and 19F, will be utilized to investigate if the bacterial loa of a serotype dominates the NP biofilm consortium during colonization and/or persistence. Viable counts by plating and fluorescence activated cell sorting (FACS) analyses will be performed. Biofilm consortiums will be imaged by microscopy. PD is produced by one strain even when some children are colonized by two serotypes. Aim#1 studies will allow us to find out whether persistence of two strains in the nasopharynx generates invasive Sp of a specific serotype. In nature, NP colonization by two strains should be sequential thereby Aim#1 studies will also investigate if pre-established NP biofilms can cohabit with, or be displaced by, the introduction of a new serotype. Aim#1 studies will be helpful in design of future vaccines. In preliminary studies, incubating two antibiotic-marked Sp serotypes generated double antibiotic resistant recombinant strains. Aim#2 will begin studying the mechanism of recombination of biofilm consortiums. Recombination frequency of two different triple-marked serotypes incubated in our well-established biofilm reactor with human respiratory cells will be obtained by plating with a selected antibiotic. Whether NP recombination occur by transformation will be investigated utilizing triple- marked mutants in the comEC gene which has been implicated in transformation. Analysis of 2 nonselected markers will indicate whether one strain serves preferentially as donor in these crosses. The results of Aim#2 will indicate the importance of recombination in the generation of multi-drug resistant strains and possibly of new serological types of Sp. This knowledge will be very helpful in controlling future disease spread. The information generated in the above proposed studies will be very relevant for human health.